A synthetic metastatic niche reveals antitumor neutrophils drive breast cancer metastatic dormancy in the lungs

Biomaterial scaffolds mimicking the environment in metastatic organs can deconstruct complex signals and facilitate the study of cancer progression and metastasis. Here we report that a subcutaneous scaffold implant in mouse models of metastatic breast cancer in female mice recruits lung-tropic circulating tumor cells yet suppresses their growth through potent in situ antitumor immunity. In contrast, the lung, the endogenous metastatic organ for these models, develops lethal metastases in aggressive breast cancer, with less aggressive tumor models developing dormant lungs suppressing tumor growth. Our study reveals multifaceted roles of neutrophils in regulating metastasis. Breast cancer-educated neutrophils infiltrate the scaffold implants and lungs, secreting the same signal to attract lung-tropic circulating tumor cells. Second, antitumor and pro-tumor neutrophils are selectively recruited to the dormant scaffolds and lungs, respectively, responding to distinct groups of chemoattractants to establish activated or suppressive immune environments that direct different fates of cancer cells.

. Influences of Gr1 + cells derived from diseased scaffolds and lungs of BALB/c mice bearing 4T1 tumor at day 14 or day 21 after tumor inoculation on suppressing in vitro proliferation of naïve T cells. Data are shown as Mean ± SEM and p values are from student's two-tailed t test (day 21 vs. day 14). n = 3.

Fig. S2.
Representative density plots for tdTomato + 4T1 cancer cells migrating from the primary tumor to scaffold implants with different sizes in BALB/c mice bearing 4T1-Luc2-tdTomato tumor. Tissues were retrieved from mice two weeks after orthotopic inoculation of tumor cells, prepared to single cell suspensions, and then measured by flow cytometer. Splenocytes and primary tumor cells were negative and positive controls, respectively.

Fig. S3.
Representative density plots for tdTomato + 4T1 cancer cells migrating to scaffold implants and lungs in tumor-free healthy BALB/c mice or diseased BALB/c mice bearing non-fluorescent 4T1 tumor. 4T1-Luc2-tdTomato cells were administrated through intracardiac injection. Figure S4. Expression of S100a8 and S100a9 genes in cell subsets of diseased lungs derived from 4T1bearing mice. Cells were isolated by magnetic-activated cell sorting and the gene expression was analyzed by qRT-PCR. EC: endothelial cell. Data are shown as Mean ± SEM. n = 3.  S5. Images of 4T1 cells transmigrating towards the S100A8/A9 recombinant proteins or conditioned media (CM) without or with supplementing anti-S100A8/A9 antibodies (Ab). Scale bar = 150 µm.  S6. Frequencies of Gr1 + CD11b + Ly6G + Ly6Cgranulocytic (neutrophils) and Gr1 + CD11b + Ly6G -Ly6C + monocytic myeloid cells in the diseased lungs and scaffolds of 4T1-bearing BALB/c mice after mice were administrated with anti-Gr1 antibodies (Ab). Data are shown as Mean ± SEM and p values are from student's two-tailed t test. n =3.   . Survival rates of scaffold-free and scaffold-bearing BALB/c mice after 4T1 primary tumors were resected at day 10, day 15, or day 20 after orthotopic tumor inoculation. p values are from Mantel-Cox test. n = 7 for groups receiving tumor resection surgery at day 10 or day 20 after tumor inoculation. n = 8 for groups receiving tumor resection surgery at day 15 after tumor inoculation.

Fig. S10
. Flow cytometric gating strategies to identify different immune subsets. There were three steps to identify the cell subsets shown in Fig. 4a. First (step 1), after identifying cell population with FSC and SSC, we used CD45 antibodies to distinguish CD45 + immune cells from non-immune cells. Second (step 2), we employed surface markers to distinguish different immune cell populations among the CD45 + population, including neutrophils, macrophages, dendritic cells, NK cells, CD8 + T cells, and CD4 + T cells. Last (step 3), for each immune cell population, we further employed surface or intracellular markers to distinguish cell subsets with different phenotypes. These markers were attached with fluorophores, including BV510, FITC/AF488, PE, PE-Cy7, or APC. We prepared unstained and single stained samples as controls to distinguish marker-positive and marker-negative populations in every flow cytometric experiment. For example, to identify the frequency of N1 neutrophils, after gating cell population according to FSC and SSC, we first (step 1) gated CD45 + immune cells with AF700-CD45 antibodies, and then (step 2) among the immune cells, we gated Gr1 + CD11b + Ly6G + neutrophils with Pacific Blue-Gr1, BV510-CD11b, and PE-Ly6G antibodies, and last (step 3) among the neutrophils, we gated NOS2 + TNFα + N1 neutrophils with PE-Cy7-NOS2 and FITC-TNFα antibodies. Details are in the Method section.    S13. The Cxcl1-to-S100a8 gene expression ratio in diseased lungs and scaffolds of 4T1-bearing BALB/c mice as a function of time after tumor inoculation. Data are shown as Mean ± SEM and p values are from student's two-tailed t test. n = 3.

Fig. S14.
Kaplan-Meier curves displaying the survival probability of breast cancer patients with low (black) or high (red) expression of genes for N1 chemoattractants (Cxcl1, Cxcl2, Cxcl5) or genes for N2 chemoattractants (S100a8, S100a9). n = number of patients with available clinical data. HR: hazard ratio. p values are from Mantel-Cox test. Table S1. Microbeads or kits used in magnetic-activated cell sorting to isolate different cell populations.